- Level 0: Ran gel of PDF, PR, and GST pGEM colonies to detect the presence of the insert (amplified in PCR using only promoter plasmids). Absolutely nothing on gel - something went wrong.
- pGEM: Miniprepped pGEM PDF, GST, and PR plasmids and measured concentrations.
- Performed level 0 digest overnight and ‘Big G’ transformed and grew overnight.
- Grew them overnight to screen for blue/white colonies in the morning.
- Investigated successful plates from second pGEM transformations. Blue and white colonies were observed on TSH, TSHH and negative LB, AMP, IPTG plates. White colonies were extracted and grew at 37 degrees for three hours (using 300 ul LB broth + AMP and colony).
- Colony PCR of controls, TSH and TSHH transformant colonies
- Organised Skype with Valencia
- Transformed agrobacteria with miniprepped B1 and B2 (and negative control).
- Protocol: add 5 microlitres of B1 and B2 to the agrobacterium, leave on ice for 10 minutes, add to liquid nitrogen for 5 minutes, 5 minutes at 37 degrees celsius, and then on ice for 5 minutes. Then add 1 ml LB (no antibiotic), shake at 28 degrees for 2 hours, spin down at 13000 rpm for 1 minute, resuspend pellet and plate onto lk-rif plates, grow overnight at 28 degrees.
- Mini prepped the P6-18J grown overnight.
- Removed coomassie stain from the gel and used the destaining agent.